It has become increasingly clear that understanding morphogenesis and disease requires three-dimensional tissue cultures and models. Effective 3D imaging techniques, capable of reporting on subcellular as well as multicellular scales, in a time-resolved manner, are crucial for achieving this goal. While the light microscope has been the main tool of investigation in biomedicine for four centuries, the current requirements for 3D imaging pose new, difficult challenges. Due to their insignificant absorption in the visible spectrum, most living cells exhibit very low contrast when imaged by visible light microscopy. Consequently, fluorescence microscopy has become the main tool of investigation in cell biology. Due to extraordinary progress in designing fluorescence tags, structures in the cell can be imaged with high specificity.
More recently, super-resolution microscopy methods based on fluorescence have opened new directions of investigation, toward the nanoscale subcellular structure. However, fluorescence imaging is subject to several limitations. Absorption of the excitation light may cause the fluorophore to irreversibly alter its molecular structure and stop fluorescing. This process, known as photobleaching, limits the time interval over which continuous imaging can be performed. The excitation light is typically toxic to cells, a phenomenon referred to as phototoxicity.
Addressing cell cultures in particular, transmitted light modalities appear to be ideal for studying cell growth and proliferation due to low phototoxicity, an absence of photobleaching and easy sample preparation. Yet, such assays are most frequently conducted with the aid of labels. While specificity granted by external markers is crucial for certain applications, quantifying cell growth over longer time scales remains a grand challenge. It has been known for some time that indicators of cell proliferation do not have equal growth. More recent methods using vibrating hollow cantilevers to weigh cell passing through are limited to non-adherent cells. Methods based on vibrating pedestals have also been demonstrated, but at the expense of mass sensitivity.
Label-free microscopy provides a solution to overcoming these limitations. Two classical methods are phase contrast (PC) microscopy and differential interference contrast (DIC) microscopy. Both PC and DIC microscopy indicate modifications to the wavefront of incident light propagating through a sample. However, to date, neither of them has provided a quantitative measure of wavefront deformation in three dimensions.
Along these lines, Cogswell et al., “Quantitative DIC microscopy using a geometric phase shifter”, Proc. SPIE 2984, Three-Dimensional Microscopy: Image Acquisition and Processing IV, (1997) proposed DIC with geometrically-induced phase shifting, applied for two-dimensional (2D) imaging. DIC with two orthogonal shear directions has been used to obtain 2D quantitative phase images, as described, for example by King et al., “Quantitative phase microscopy through differential interference imaging,” Journal of Biomedical Optics, vol. 13(2), 024020 (2008). Both of these references are incorporated herein by reference.
Quantitative phase imaging (QPI) is an approach that quantitatively ascertains the phase shift in a wavefront propagating through a refractive medium such as a cell. QPI is described in Popescu, Quantitative Phase Imaging of Cells and Tissues, (McGraw-Hill, 2011), which is incorporated herein by reference. However, imaging optically thick, multiple scattering specimens is challenging for any optical method, including QPI. The fundamental obstacle is that multiple scattering generates an incoherent background, which ultimately degrades the image contrast. An imaging method dedicated to imaging optically thick specimens must include a mechanism to subdue the multiple scattering backgrounds and exhibit strong spatial sectioning to suppress the out of focus light.
The invention described below in directed toward solving the problem of quantitatively measuring phase shift as a function of position in three-dimensions within a sample, even if it is optically thick and gives rise to multiple scattering.